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Notes: pCAT promoter constructs were used to evaluate the effects of IL-10 and IGF-I on MMP-2 expression.The CAT assays were performed using the CAT Enzyme Assay System with Reporter Lysis Buffer. (2637)

Notes: Total RNA was isolated from TOP10F’ or INVaF' E. coli (Invitrogen) using the SV Total RNA Isolation System. The isolated RNA was used in RT-PCR to detect cleavage of marA mRNA. A marA drug resistance gene sequence was also ligated into a chloramphenicol acetyltransferase (CAT) reporter vector. This construct was used to measure single-stranded nanovector-transcribed ribozyme activity on the marA sequence. CAT activity was measured using the CAT Enzyme Assay System. CAT activity was normalized by cotransfecting cells with the pSV-β-Galactosidase Vector and assaying lysates with the β-Galactosidase Enzyme Assay System. (2794)

Notes: The cloned 752 amino acid GC-binding factor 2 (GCF2) was expressed in vitro using the TNT® Coupled Reticulocyte Lysate System. The synthesized product migrated at ~160kDa by SDS-PAGE when the predicted molecular weight was 83kDa. Expression of the protein from E.coli also produced a protein that migrated at ~160kDa. Mass spectrum analysis confirm a mass of 83kDa. The E.coli produced protein was further characterized by gel shift analysis with the AP-2 Concensus Oligonucleotide. Recombinant GCF2, AP-2 and SP1 transcription factors were analyzed for binding to the EGF receptor promoter by footprint analysis. The GCF2 protein was expressed with a EGF Receptor promoter-CAT reporter vector in CV-1 cells and found to increase CAT activity. (0510)

Notes: To assess the ability of a cytoplasmic Semliki Forest virus (SFv) expression system to generate an RNA for packaging containing an intron, the promoter, intron, CAT gene and poly A signal of the pCAT® Control Vector was inserted into a SFv vector. The SFv sequences and the CAT sequences were transcribed in vitro and co-transfected into BHK-21 cells with SFv vectors containing the Moloney murine leukemia virus packaging proteins. This method produced retrovirus particles that were of high titer and were used to infect NIH 3T3 cells. The CAT gene expression in the NIH 3T3 cells was measured with the CAT Enzyme Assay System. The presence of the intron in the infected cells was confirmed by isolating total RNA and performing RT-PCR with primers that could distinguish whether or not the cells got the CAT gene with the intron. RT-PCR was performed with the Access RT-PCR System. (0795)

J. Biol. Chem.272, 20756-20763.
Characterization of point mutations in patients with X-linked ichthyosis. Effects On the structure and function of the steroid sulfatase protein.1997

Alperin, E. S. and Shapiro, L. J.

Notes: Transcripts were produced using the RiboMAX™ Large Scale RNA Production System and expressed in the Rabbit Reticulocyte Lysate System with or without Canine Pancreatic Microsomal Membranes (CMMs). Proteinase K protection assays were performed with proteins expressed in the presence of the membranes with and without Triton® X-100 solubilization of the membranes. (1509)

J. Biol. Chem.272(33), 20756-20763.
Characterization of point mutations in patients with X-linked ichthyosis: Effects on the structure and function of the steroid sulfatase protein.1997

Alperin, E.S. and Shapiro, L.J.

Notes: The ribonuclease inhibitor was used to protect RNA during the reverse transcriptase reaction for RT-PCR. The RNasin® Ribonuclease Inhibitor is also used in the RiboMAX® System for RNA stabilization during in vitro transcription. Transcripts were produce with the RiboMAX® System and expresed in Rabbit Lysate with or without Canine Microsomal Membranes. Proteinase K protection assays are performed with proteins expressed in the presence of the membranes with and without Triton X-100 solubilization of the membranes. The proteinase K digests were stopped by the addition of PMSF and immediately loading the sample into 100°C SDS Sample buffer. (1670)

J. Biol. Chem.272, 3674-3682..
Expression of the 90K immunostimulator gene is controlled by a promoter with unique features.1997

Brakebusch, C., Jallal, B., Fusco, O., Iacobelli, S., Ullrich, A.

Notes: CAT reporter studies were performed in NIH3T3 cells using the pCAT® Basic Vector, pCAT® Promoter Vector and the CAT Enzyme Assay System. (Promega's pCAT Vectors have now been replaced with the improved pCAT®-3 Vector series). (1399)

Notes: Mutant and wildtype arginine vasopressin (AVP) constructs were co-transfected with a CAT-expression vector containing a neo resistance gene. Stable transformants of Neuro2A cells were generated with the aid of the Transfectam® Reagent and G418 sulfate. CAT activity in stably transfected clones was determined with the CAT Enzyme Assay System. The effect of the mutant AVP constructs on cell viability was determined with the CellTiter 96® AQueous Cell Proliferation Assay (MTS/PMS). Several constructs displayed marked decreases in cell viability. The cause was necrosis rather than apoptosis due to the absence of DNA laddering and a negative reaction in the Apoptosis Detection System, Fluoroscein (data not shown). The name of the Apoptosis Detection System, Fluorescein, has changed to DeadEnd™ Fluorometric TUNEL System. (1009)

Notes: CAT reporter activity was normalized to luciferase activity using the Luciferase Assay System. Luciferase was provided by the pGL3 Control Vector and CAT activity was measured using the CAT Enzyme Assay System. (1237)

Notes: The pCAT® Basic Vector was used to construct recombinant Adenovirus reporter vectors. The recombinant virii were used to study promoter activity in the pancreatic cell line AR-42J. Cells were lysed with Reporter Lysis Buffer for assay. (1208)

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